Project summary/Abstract This proposal explores the role of nuclear O-fucose as a novel regulatory mechanism in Toxoplasma gondii, cause of severe neurological and ocular diseases. The John Samuelson lab used the anti-fucose Aleuria aurantia lectin (AAL) to show that fucosylated proteins of T. gondii form punctate assemblies in close association with the nuclear pore complex. The assemblies of O-fucosylated proteins are reminiscent of Cajal, PML, and histone locus bodies in the host nucleus, which are not modified by O-fucose. AAL-enrichment and mass spectrometry showed O-fucose is attached to Ser and Thr in intrinsically disordered regions of dozens of proteins, which appear to be involved in transcription, mRNA processing and transport, and signaling. The Chris West lab identified the T. gondii O-transferase (TgSpy), which contains 11 N-terminal tetratricopeptide repeats (TPRs) and a C-terminal GT41 glycosyltransferase domain. Recombinant TgSpy made in the cytoplasm of bacteria hydrolyses GDP-fucose and O-fucosylates itself, a polySer/GST fusion, and proteins in lysates from spyKO cells. TgSpy is not essential, but the spyKO grows more slowly, while transfectants that overexpress TgSpy grow faster. Green fluorescent protein fused with polySer is O-fucosylated and accumulates in the AAL- labeled assemblies in the parent strain but is degraded in the spyKO. TgSpy is a homolog of plant Spindly, a negative regulator of gibberellic acid signaling in Arabidopsis, and is also a paralog the host O-GlcNAc transferase (OGT). The OGT, which is absent in Tg, modifies intrinsically disordered regions of hundreds of proteins and is an important factor in stress resistance and nutrient signaling. According to precedent provided by OGT, we hypothesize that TgSpy modifies proteins and affects their activity under normal growth conditions and in response to stress. In addition, modification by TgSpy uniquely causes O-fucosylated proteins to localize near the NPC, where they are protected from degradation. The Samuelson and West labs will work together to dissect how O-fucose works in T. gondii. In Aim 1 we will evaluate the importance of the TPRs and GT41 glycosyltransferase for O-fucosylation and parasite growth. In Aim 2 we will determine the stability, localization, and function of five proteins, which are O-fucosylated and contribute to Tg fitness, in response to varied Spy expression. We will perform interactome studies to address the mechanism of protein inclusion in assemblies of O-fucosylated proteins and pulse-chase labeling, time- lapse microscopy, and photo-bleaching to determine the stability of assemblies. In Aim 3 we will compare the growth in culture, mice, and cats of the parental strain, the spy knockout, and organisms overexpressing TgSpy. In addition, we will perform RNA-seq and SILAC on the three sets of protists growing as tachyzoites or converting to bradyzoites in culture.